Dicty News Electronic Edition Volume 13, number 9 October 23, 1999 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ======================== Need Dicty 99 Pictures ======================== We are looking for pictures from the Bar Harbor Dicty meeting. If you have pictures you are willing to share, please email them to dicty@nwu.edu. If you don't have access to a scanner, contact us--you can send us the picture, we'll scan it and return the photo to you. ============= Abstracts ============= Ca2+/calmodulin-independent activation of calcineurin from Dictyostelium by unsaturated long chain fatty acids Ursula Kessen, Ralph Schaloske, Annette Aichem, and Rupert Mutzel Fakultaet für Biologie, Universitaet Konstanz, D-78457 Konstanz, Germany J. Biol. Chem., in press Summary This study describes a novel mode of activation for the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. Using purified calcineurin from Dictyostelium discoideum we found a reversible, Ca2+/calmodulin independent activation by the long chain unsaturated fatty acids arachidonic acid, linoleic acid and oleic acid which was of the same magnitude as activation by Ca2+/calmodulin. Half maximal stimulation of calcineurin occurred at fatty acid concentrations of approximately 10 microM with either p-nitrophenyl phosphate or RII phosphopeptide as substrates. The methyl ester of arachidonic acid and the saturated fatty acids palmitic acid and arachidic acid did not activate calcineurin. The activation was shown to be independent of the regulatory subunit, calcineurin B. Activation by Ca2+/calmodulin and fatty acids was not additive. In binding assays with immobilized calmodulin, arachidonic acid inhibited binding of calcineurin to calmodulin. Therefore fatty acids appear to mimic Ca2+/calmodulin action by binding to the calmodulin-binding site. ---------------------------------------------------------------------------- Reconstitution of Membrane Transport Powered by a Novel Dimeric Kinesin Motor of the Unc104/KIF1A Family Purified from Dictyostelium Nira Pollock*, Eugenio L. de Hostos^, Christoph W. Turck#, and Ronald D. Vale#* *Departments of Cellular and Molecular Pharmacology, ^Pathology and #The Howard Hughes Medical Institute, University of California, San Francisco, CA Journal of Cell Biology, in press Abstract Motor-powered movement along microtubule tracks is important for membrane organization and trafficking. However, the molecular basis for membrane transport is poorly understood, in part because of the difficulty in reconstituting this process from purified components. Using video microscopic observation of organelle transport in vitro as an assay, we have purified two polypeptides (245 kDa and 170 kDa) from Dictyostelium extracts that independently reconstitute plus-end-directed membrane movement at in vivo velocities. Both polypeptides were found to be kinesin motors, and the 245 kDa protein (called DdUnc104) is a close relative of C. elegans Unc104 and mouse KIF1A, neuron-specific motors that deliver synaptic vesicle precursors to nerve terminals. A knockout of the DdUnc104 gene produces a pronounced defect in organelle transport in vivo and in the reconstituted assay. Interestingly, DdUnc104 functions as a dimeric motor, in contrast to other members of this kinesin subfamily which are monomeric. ---------------------------------------------------------------------------- Commentary Slime molds, ascidians, and the utility of evolutionary theory Leo W. Buss Departments of Ecology & Evolutionary Biology and Geology & Geophysics, Yale University, New Haven, CT 06520-8106 PNAS. Vol. 96, Issue 16, 8801-8803, August 3, 1999 This is a commentary on the interest of Dictyostelium to evolutionary biology. There is no abstract but the full text can be found at: http://www.pnas.org/cgi/content/full/96/16/8801 ---------------------------------------------------------------------------- DYNEIN MOTOR REGULATION STABILIZES INTERPHASE MICROTUBULE ARRAYS AND DETERMINES CENTROSOME POSITION. Michael P. Koonce 1, Jana Köhler 2, Ralph Neujahr 2, Jean-Marc Schwartz 2, Irina Tikhonenko 1, and Günther Gerisch 2. 1 Division of Molecular Medicine, Wadsworth Center, Empire State Plaza, Albany, New York. 12201-0509. 2 Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany EMBO Journal, in press ABSTRACT Cytoplasmic dynein is a microtubule-based motor protein responsible for vesicle movement and spindle orientation in eukaryotic cells. We show here that dynein also supports microtubule architecture and determines centrosome position in interphase cells. Overexpression of the motor domain in Dictyostelium leads to a collapse of the interphase microtubule array, forming loose bundles that often enwrap the nucleus. Using GFP-a-tubulin to visualize microtubules in live cells, we show that the collapsed arrays remain associated with centrosomes and are highly motile, often circulating along the inner surface of the cell cortex. This is strikingly different from wild type cells where centrosome movement is constrained by a balance of tension on the microtubule array. Centrosome motility involves force-generating microtubule interactions at the cortex, with the rate and direction consistent with a dynein-mediated mechanism. Mapping the overexpression effect to a carboxy-terminal region of the heavy chain highlights a functional domain within the massive sequence important for regulating motor activity. ---------------------------------------------------------------------------- Identification of a UDP-GlcNAc:Skp1-Hydroxyproline GlcNAc-Transferase in the Cytoplasm of Dictyostelium Patana Teng-umnuay, Hanke van der Wel, and Christopher M. West Department of Anatomy & Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610-0235, USA J. Biol. Chem., in press Summary: Skp1 is a cytoplasmic and nuclear protein required for the ubiquitination of cell cycle regulatory proteins and transcriptional factors. In Dictyostelium, Skp1 is modified by a linear pentasaccharide, Gala1-6Gala1-Fuca1-2Galb1-3GlcNAc, attached to a HyPro residue at position 143. To study the formation of the GlcNAc-HyPro linkage, an assay was developed for the transfer of [3H]GlcNAc from UDP-[3H]GlcNAc to Skp1-HyPro143 or a synthetic Skp1 4-HyPro-peptide. The cytosolic but not the particulate fraction of the cell mediated transfer in a time-, concentration-, and HyPro-dependent fashion. Incorporated radioactivity was alkali-resistant and was recovered as GlcNH2 after acid hydrolysis, consistent with linkage of GlcNAc to HyPro. The GlcNAc-transferase activity was purified 130,000-fold as a single component with a recovery of 5%. Key to the purification was the synthesis of a novel affinity resin linking UDP-GlcNAc at its 5-uridyl position. The purified activity had an apparent Mr of ~45,000 by gel filtration, required DTT and a divalent cation, and consisted predominantly of a Mr 51,000 band after SDS-PAGE which was photoaffinity-labeled with 5-[125I]ASA-UDP-GlcNAc in a UDP-GlcNAc-sensitive fashion. Its apparent Km's for UDP-GlcNAc and Skp1 were submicromolar. The presence of the enzyme in the cytosolic fraction, its dependence on a reducing environment, and its high affinity for UDP-GlcNAc, strongly suggest that Skp1 is glycosylated by a HyPro GlcNAc-transferase which resides in the cytoplasm. ---------------------------------------------------------------------------- A Linking Function for the Cellulose-Binding Protein SP85 in the Spore Coat of Dictyostelium discoideum Yunyan Zhang, Ping Zhang and Christopher M. West Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610-0235 USA J. Cell Sci., in press Summary: SP85 is a multidomain protein of the Dictyostelium spore coat whose C-terminal region binds cellulose in vitro. To map domains critical for localizing SP85 and for binding to other proteins in vivo, its N- and C-terminal regions, and a hybrid fusion of the N- and C-regions, were expressed in prespore cells. Immunofluorescence showed that only the N-terminal region and the N/C-hybrid accumulated in prespore vesicles, where coat proteins are normally stored prior to secretion. In contrast, only the C-terminal region and N/C-hybrid were incorporated into the coat after secretion. To determine if SP85 is important for the incorporation of other coat proteins, an SP85-null strain was created and found to mislocalize the coat protein SP65 to the interspore matrix. In vitro binding studies demonstrated that the SP85 C-terminal region bound SP65 and cellulose simultaneously, and SP65 incorporation was rescued in vivo by the C-terminal region. SP85-null spores showed increased latent permeability to a fluorescent lectin probe and accelerated germination times, and decreased bouyant density of their coats, suggesting that coat barrier functions were compromised. Dominant negative reductions in barrier functions also resulted from expression of the SP85 terminal regions, suggesting that a linking activity was important for SP85's function. Thus, separate domains of SP85 specify prespore vesicle compartmentalization and coat incorporation, and additional domains link SP65 to the coat and simultaneously interact with other binding partners which contribute to coat barrier functions. ---------------------------------------------------------------------------- [End Dicty News, volume 13, number 9]